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- EMDB-30545: Cryo-EM structure of Schizosaccharomyces pombe Atg9 of trimer -

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Basic information

Entry
Database: EMDB / ID: EMD-30545
TitleCryo-EM structure of Schizosaccharomyces pombe Atg9 of trimer
Map data
SampleProtein detergent complex (Atg9-LMNG ) treated with GraFix:
Atg9
Function / homology
Function and homology information


late nucleophagy / protein localization to phagophore assembly site / phagophore assembly site membrane / fungal-type vacuole membrane / phagophore assembly site / autophagy of mitochondrion / autophagosome assembly / autophagosome / macroautophagy / autophagy ...late nucleophagy / protein localization to phagophore assembly site / phagophore assembly site membrane / fungal-type vacuole membrane / phagophore assembly site / autophagy of mitochondrion / autophagosome assembly / autophagosome / macroautophagy / autophagy / protein transport / cytoplasmic vesicle membrane / Golgi membrane / endoplasmic reticulum membrane / integral component of membrane
Autophagy-related protein 9
Autophagy-related protein 9
Biological speciesSchizosaccharomyces pombe (fission yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsMatoba K / Tsutsumi A
Funding support Japan, 9 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)18K06097 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101001 (support number 0053) Japan
Japan Society for the Promotion of Science (JSPS)15K21608 Japan
Japan Science and TechnologyJPMJCR14M1 Japan
Japan Science and TechnologyJPMJCR13M7 Japan
Japan Society for the Promotion of Science (JSPS)25111004 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101079 (support number 0739) Japan
Japan Society for the Promotion of Science (JSPS)19H05707 Japan
Japan Society for the Promotion of Science (JSPS)18H03989 Japan
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion.
Authors: Kazuaki Matoba / Tetsuya Kotani / Akihisa Tsutsumi / Takuma Tsuji / Takaharu Mori / Daisuke Noshiro / Yuji Sugita / Norimichi Nomura / So Iwata / Yoshinori Ohsumi / Toyoshi Fujimoto / ...Authors: Kazuaki Matoba / Tetsuya Kotani / Akihisa Tsutsumi / Takuma Tsuji / Takaharu Mori / Daisuke Noshiro / Yuji Sugita / Norimichi Nomura / So Iwata / Yoshinori Ohsumi / Toyoshi Fujimoto / Hitoshi Nakatogawa / Masahide Kikkawa / Nobuo N Noda /
Abstract: The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM) ...The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.
History
DepositionSep 14, 2020-
Header (metadata) releaseOct 28, 2020-
Map releaseOct 28, 2020-
UpdateOct 28, 2020-
Current statusOct 28, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0291
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0291
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_30545.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.55 Å/pix.
x 140 pix.
= 216.3 Å
1.55 Å/pix.
x 140 pix.
= 216.3 Å
1.55 Å/pix.
x 140 pix.
= 216.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.545 Å
Density
Contour LevelBy AUTHOR: 0.0291 / Movie #1: 0.0291
Minimum - Maximum-0.022595631 - 0.07088186
Average (Standard dev.)0.0018120831 (±0.005914598)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 216.29999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.5451.5451.545
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z216.300216.300216.300
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.0230.0710.002

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Supplemental data

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Segmentation: #1

Fileemd_30545_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_30545_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_30545_half_map_2.map
Projections & Slices
AxesZYX

Projections

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Density Histograms

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Sample components

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Entire Protein detergent complex (Atg9-LMNG ) treated with GraFix

EntireName: Protein detergent complex (Atg9-LMNG ) treated with GraFix
Number of components: 2

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Component #1: cellular-component, Protein detergent complex (Atg9-LMNG ) treate...

Cellular-componentName: Protein detergent complex (Atg9-LMNG ) treated with GraFix
Recombinant expression: No
SourceSpecies: Schizosaccharomyces pombe (fission yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast) / Vector: pRS426 / Strain: BJ926

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Component #2: protein, Atg9

ProteinName: Atg9 / Recombinant expression: No
SourceSpecies: Schizosaccharomyces pombe (fission yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/mL / pH: 8.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 279 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 2000.0 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 43113
3D reconstructionSoftware: RELION / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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